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Project description:Shroom3-Fyn interaction determines nephrin phosphorylation and Albuminuria in allografts
| PRJNA432731 | ENA
Project description:BACKGROUND:We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS:We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. RESULTS:Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. CONCLUSIONS:We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.
| S-EPMC6218856 | biostudies-literature
Project description:We investigated the clinical and molecular significance of minimal peritubular capillary (PTC) and isolated glomerular C4d+ staining using microarrays. Immunohistochemistry for C4d was performed on paraffin-embedded sections. Of the 255 biopsies analyzed, 51% were C4d negative, 4% were minimal, 15% focal or diffuse PTC C4d+, and 31% isolated glomerular C4d+. Minimal and focal/ diffuse PTC C4d+ staining were associated with a higher frequency of donor-specific anti-HLA antibodies (DSA) (67% vs. 82% vs. 25%), antibody mediated rejection (AMR) (66% vs. 89% vs. 19%) and mean glomerulitis (0.88 vs. 0.65 vs. 0.25, p=0.003), interstitial inflammation (1.25 vs. 1.41 vs. 0.79; p=0.003) and peritubular capillaritis scores (1.5 vs. 1.5 vs. 0.34; p < 0.001), compared to the C4d negative group, respectively. There were no differences in the DSA frequency, AMR rate, and Banff scores between isolated glomerular C4d+ and negative patients. While both minimal and focal/diffuse C4d+ biopsies showed increased expression of genes related to the immune response, and interferon-gamma and rejection induced, cytotoxic T cell and constitutive macrophage-associated pathogenesis based transcripts, there was no activation of immune-response related genes in isolated glomerular C4d+ biopsies. In summary, minimal PTC C4d+ staining but not isolated glomerular C4d+ staining is associated with AMR.
2012-05-29 | GSE38262 | GEO
Project description:We investigated the clinical and molecular significance of minimal peritubular capillary (PTC) and isolated glomerular C4d+ staining using microarrays. Immunohistochemistry for C4d was performed on paraffin-embedded sections. Of the 255 biopsies analyzed, 51% were C4d negative, 4% were minimal, 15% focal or diffuse PTC C4d+, and 31% isolated glomerular C4d+. Minimal and focal/ diffuse PTC C4d+ staining were associated with a higher frequency of donor-specific anti-HLA antibodies (DSA) (67% vs. 82% vs. 25%), antibody mediated rejection (AMR) (66% vs. 89% vs. 19%) and mean glomerulitis (0.88 vs. 0.65 vs. 0.25, p=0.003), interstitial inflammation (1.25 vs. 1.41 vs. 0.79; p=0.003) and peritubular capillaritis scores (1.5 vs. 1.5 vs. 0.34; p < 0.001), compared to the C4d negative group, respectively. There were no differences in the DSA frequency, AMR rate, and Banff scores between isolated glomerular C4d+ and negative patients. While both minimal and focal/diffuse C4d+ biopsies showed increased expression of genes related to the immune response, and interferon-gamma and rejection induced, cytotoxic T cell and constitutive macrophage-associated pathogenesis based transcripts, there was no activation of immune-response related genes in isolated glomerular C4d+ biopsies. In summary, minimal PTC C4d+ staining but not isolated glomerular C4d+ staining is associated with AMR. Total of 92 arrays included in this study. G1. Native pre-implantation biopsies (N= 10); G2. Focal or diffuse PTC C4d+ (N= 13); G3. Minimal PTC C4d+ (N=4); G4. Isolated glomerular C4d+ with glomerular disease (N= 13); G5. Isolated glomerular C4d+ staining without glomerular disease (N= 15); G6. C4d negative with glomerular disease (N= 12); G7. C4d negative biopsies without evidence of glomerular disease (N=25)
2012-05-28 | E-GEOD-38262 | biostudies-arrayexpress
Project description:<p>Naïve human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naïve hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naïve hESCs, indicating that PRODH maintains naïve pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naïve pluripotency of hESCs.</p><p><br></p><p><strong>Targeted metabolomics</strong> (amino acids and derivatives) - H9P (Primed) VS H9N (Naïve) - is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7832' rel='noopener noreferrer' target='_blank'><strong>MTBLS7832</strong></a>.</p><p><strong>Untargeted metabolomics</strong> - H9N PLKO.1 vs H9P PLKO.1 / H9P shPRODH vs H9P PLKO.1 / H9N shPRODH vs H9N PLKO.1 - is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7840' rel='noopener noreferrer' target='_blank'><strong>MTBLS7840</strong></a>.</p>
2024-02-12 | MTBLS7832 | MetaboLights
Project description:<p>Naïve human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naïve hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naïve hESCs, indicating that PRODH maintains naïve pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naïve pluripotency of hESCs.</p><p><br></p><p><strong>Untargeted metabolomics</strong> - H9N PLKO.1 vs H9P PLKO.1 / H9P shPRODH vs H9P PLKO.1 / H9N shPRODH vs H9N PLKO.1 - is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7840' rel='noopener noreferrer' target='_blank'><strong>MTBLS7840</strong></a>.</p><p><strong>Targeted metabolomics</strong> (amino acids and derivatives) - H9P (Primed) VS H9N (Naïve) - is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7832' rel='noopener noreferrer' target='_blank'><strong>MTBLS7832</strong></a>.</p>
2024-02-12 | MTBLS7840 | MetaboLights
Project description:Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft losses. AMR is caused by donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium, which together with high interferon gamma (IFNɣ) and tumor necrosis factor-alpha (TNFα), trigger graft injury. Unfortunately, the mechanisms governing cell-specific injury in AMR remain unclear. We studied 30 for-cause kidney biopsies with early AMR, acute cellular rejection or acute tubular necrosis (‘non-AMR’). We laser-captured microdissected glomeruli and tubulointerstitium and subjected them to unbiased proteome analysis. 120/2026 glomerular and 180/2399 tubulointerstitial proteins were significantly differentially expressed in AMR vs. non-AMR biopsies (p<0.05). Basement membrane and extracellular matrix (ECM) proteins were significantly decreased in both AMR compartments. We verified decreased glomerular and tubulointerstitial LAMC1 expression, and decreased glomerular NPHS1 and PTPRO expression in AMR. Cathepsin-V (CTSV) was predicted to cleave ECM-proteins in the AMR glomeruli, and CTSL, CTSS and LGMN in the tubulointerstitium. We identified galectin-1, an immunomodulatory protein upregulated in AMR glomeruli and linked to the ECM. Anti-HLA class-I antibodies significantly increased CTSV expression, and galectin-1 expression and secretion, in human glomerular endothelial cells. We also studied glutathione S-transferase omega-1 (GSTO1), an ECM-modifying enzyme, increased in the AMR tubulointerstitium. GSTO1 expression was significantly increased in TNFα-treated proximal tubular epithelial cells. IFNɣ and TNFα significantly increased CTSS and LGMN expression in these cells. Basement membranes are often remodeled in chronic AMR, and we demonstrated that this remodeling begins early in glomeruli and tubulointerstitium. Targeting ECM-remodeling in AMR may represent a new therapeutic opportunity.
2020-09-22 | PXD017580 | Pride
Project description:Calcimimetic agents allosterically increase the calcium (Ca2+) sensitivity of the calcium-sensing receptor (CaSR), which is expressed in the tubular system and to a lesser extent in podocytes. Activation of this receptor can reduce glomerular proteinuria and structural damage in proteinuric animal models. However, the precise role of the podocyte CaSR is still unclear. A CaSR knockdown in cultured murine podocytes and a podocyte-specific CaSR knockout in BALB/c mice were generated to study its role in proteinuria and kidney function. Podocyte CaSR knockdown abolished the calcimimetic R-568 mediated Ca2+-influx, disrupted the actin cytoskeleton, reduced cellular attachment and migration velocity. Adriamycin (ADR)-induced proteinuria enhanced glomerular CaSR expression in wild type mice. Albuminuria, podocyte foot process effacement, podocyte loss and glomerular sclerosis were significantly more pronounced in ADR-treated podocyte-specific CaSR knockout mice compared to wild type littermates. The co-treatment of WT mice with ADR and the calcimimetic cinacalcet reduced the proteinuria in WT, but not in podocyte specific CaSR knockout mice. In addition, four children with nephrotic syndrome, objecting glucocorticoid therapy, were treated with cinacalcet for 1 to 33 days. Proteinuria declined transiently by up to 96% and edema resolved. The activation of podocyte CaSR regulates key podocyte functions in vitro and reduces toxin induced proteinuria and glomerular damage in mice. Our findings suggest a potential novel role of CaSR signaling in control of glomerular disease. Proteomic samples: two backgrounds (CaSR Knockdown vs WT) two conditions (Treatment = 1µM R568 vs untreated control) two timepoints (24h and 48h) five replicates each 2 x 2 x 2 x 5 = 40 samples
2022-03-14 | PXD030416 | Pride
Project description:Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.
| S-EPMC6029619 | biostudies-literature
Project description:p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.
| S-EPMC4614884 | biostudies-other